Method for producing cobalt-protein complex

ABSTRACT

A method for obtaining a cobalt-apoferritin complex according to the present invention includes: the step a) of preparing a solution including a Co 2+  ion, a protein, a pH buffer agent and a Co 2+  associating agent; and the step b) of adding an oxidizing agent to the solution and thereby making the protein contain a fine particle including cobalt.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of International Patent ApplicationPCT/JP02/10127, filed Sep. 27, 2002.

BACKGROUND OF THE INVENTION

The present invention relates to a method for producing particles, andmore particularly related to a method for producing a cobalt-proteincomplex containing a cobalt particle and its related technologies.

In recent years, there have been vigorous studies in the field ofbioelectronics which is a combination of biotechnology and electronics.As a result of such studies, biosensors using proteins such as enzymes,or like devices have been already practically used.

As an attempt of the application of biotechnology to other fields, thereis a study of incorporating particles composed of a metal or a metalcompound into apoferritin which is a protein having the function ofholding a metal compound, thereby producing particles having a uniformdiameter at the nano-order level. In order to introduce various kinds ofmetals, metal compounds or the like into apoferritin according tovarious applications, many researches have been carried out.

Hereinafter, description of apoferritin will be given. Apoferritin is aprotein which is extensively present in the biological world. It has thefunction of adjusting the amount of iron which is a necessarymicronutrient element in a living body. A complex of iron or an ironcompound with apoferritin is called “ferritin”. When the amount of ironexceeds a necessary level in a living body, iron could be harmful. So,excessive iron is stored in a living body in the form of ferritin.Ferritin releases iron ions, when necessary, and then it becomesapoferritin again.

FIG. 1 is a schematic view of the structure of apoferritin. As shown inFIG. 1, apoferritin 1 is a globular protein in which 24 monomer subunitseach being composed of a polypeptide chain assemble and formnon-covalent bonds and which has a molecular weight of about 460,000.The globular protein has a diameter of about 12 nm and exhibits higherthermal stability and higher pH stability than normal proteins. Theapoferritin 1 has a hollow-like holding portion 4 having a diameter ofabout 6 nm in the center. The holding portion 4 is connected to theoutside via a channel 3. For example, when ferric iron ions areincorporated into the apoferritin 1, the iron ions enter the apoferritin1 through the channel 3 and are oxidized in a portion called theferrooxidase center in one of the subunits. Then, they reach the holdingportion 4 and finally are condensed in a negative charge region locatedin the inside surface of the holding portion 4. Then, 3000-4000 ironatoms assemble and are held in the holding portion 4 in the crystallineform of ferrihydride (5Fe₂O₃.9H₂O). A particle containing metal atomsheld in the holding portion 4 has the almost same diameter as that ofthe holding portion 4, i.e., about 6 nm.

Using apoferritin, a complex of apoferritin with particles artificiallymade to contain a metal other than iron or a metal compound is produced.

Up until today, there have been reports of introductions of metals ormetal compounds into apoferritin, such as introduction of manganese intoapoferritin (P. Mackle, 1993, J. Amer. Chem. Soc. 115, 8471-8472; F. C.Meldrum et al., 1995, J. Inorg. Biochem. 58, 59-68), introduction ofuranium into apoferritin (J. F. Hainfeld, 1992, Proc. Natil. Acad. Sci.USA 89, 11064-11068), introduction of beryllium into apoferritin (D. J.Price, 1983, J. Biol. Chem. 258, 10873-10880), introduction of aluminuminto apoferritin (J. Fleming, 1987, Proc. Natl. Acad. Sci. USA, 84,7866-7870), and introduction of zinc into apoferritin (D. Price and J.G. Joshi, Proc. Natl. Acd. Sci. USA, 1982, 79, 3116-3119). Particlescomposed of any one of these metals or metal compounds have also aboutthe same diameter as that of the holding portion 4 of apoferritin, i.e.,about 6 nm.

Processes by which a particle containing iron atoms is formed in thenatural world will be briefly described hereinafter.

On the surface of the channel 3 (see FIG. 1) connecting the outside andinside of the apoferritin 1, amino acid residues with a negative chargeat pH 7-8 are exposed and Fe²⁺ ions with a positive charge areincorporated into the channel 3 through electrostatic interaction.

Also, on the inside surface of the holding portion 4 of the apoferritin1, many glutamic acid residues which are amino acid residues and have anegative charge at pH 7-8 are exposed as on the inside surface of thechannel 3. Fe²⁺ ions incorporated through the channel 3 are oxidized atthe ferroxidase center and then are introduced to the holding portion 4located at the inside of the apoferritin 1. Iron ions are condensedthrough electrostatic interaction, and then nucleation of ferrihydride(5Fe₂O₃.9H₂O) crystals occurs.

Thereafter, increasingly incorporated iron ions are adhered to thenucleus of a ferrihydride crystal and the nucleus composed of iron oxideis grown. Thus, particles with a diameter of 6 nm are formed in theholding portion 4. This is how iron ions are incorporated and particlescomposed of iron oxide is formed.

The mechanism of incorporation of iron ions into apoferritin has beendescribed. However, since ions of any other metals which have beenreported regarding introduction thereof into apoferritin have a positivecharge, ions are incorporated into apoferritin by almost the samemechanism as that for iron ions.

SUMMARY OF THE INVENTION

As for introduction of cobalt into apoferritin, Douglas et al. havereported introduction of cobalt hydroxide (CoO(OH)) (T. Douglas and V.T. Stark, “Nanophase Cobalt Oxyhydroxide Mineral Synthesizer within theProtein Cage of Ferritin”, Inorg. Chem., 39, 2000, 1828-1830). With themethod reported by Douglas et al., a cobalt-apoferritin complexcontaining a cobalt particle can be produced.

In the method of Douglas, however, no buffer solution is used and thusthe pH of a solution in which a cobalt-apoferritin complex containing acobalt particle is formed can be changed (reduced). Specifically, if thesolution is left to stand for a few days, some of the cobalt particlescontained therein are eluted into the solution. This makes it difficultto maintain the diameter of each cobalt particle contained therein atthe diameter thereof before the operations. Therefore, it is difficultto obtain a cobalt-apoferritin complex containing a cobalt particlehaving a uniform diameter.

To solve the above-described problems, the present invention has beendevised, and it is therefore an object of the present invention toprovide a method for obtaining a cobalt-protein complex containing acobalt particle having a uniform diameter.

A method for producing a cobalt-protein complex according to the presentinvention includes: the step a) of preparing a solution containing Co²⁺ions, a protein, a pH buffer agent and a Co²⁺ associating agent; and thestep b) of adding an oxidizing agent to the solution and thereby makingthe protein contain particles composed of cobalt.

With a Co²⁺ associating agent, Co²⁺ ions are condensed in the inside ofa protein. Thus, a reaction of the Co²⁺ ions and an oxidizing agent ispreferentially occurs. At this time, by adjusting the pH of a solutionto a desired level using a pH buffer agent, the reaction of the Co²⁺ions and the oxidizing agent is prevented from proceeding in the reversedirection, and thus elution of cobalt particles contained in the proteincan be prevented. Accordingly, a cobalt-protein complex containing acobalt particle having a uniform diameter can be obtained.

It is preferable that each of the pH buffer agent and the Co²⁺associating agent is HEPES.

HEPES has functions as a pH buffer agent and the Co²⁺ associating agent.Therefore, it is not necessary to prepare a pH buffer agent and a Co²⁺associating agent separately.

The protein may be apoferritin.

The oxidizing agent may be H₂O₂.

Another method for producing a cobalt-protein complex includes: the stepa) of preparing a solution containing Co²⁺ ions, apoferritin and HEPES;and the step b) of adding H₂O₂ to the solution and thereby making theapoferritin contain particles composed of cobalt.

According to the present invention, a reaction of Co²⁺ ions and H₂O₂occurs in a HEPES solution. Thus, the pH of the solution is constant andelution of cobalt particles contained in apoferritin can be prevented.Accordingly, a cobalt-protein complex containing a cobalt particlehaving a uniform diameter can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the structure of apoferritin.

FIG. 2 is a flowchart illustrating a method for producing acobalt-apoferitin complex according to Embodiment 1.

FIG. 3 is a schematic illustration of a state of a reaction solution.

FIG. 4 is an electron micrograph of a cobalt-apoferritin complexobtained in Embodiment 1.

FIG. 5 is an electron micrograph of apoferritin obtained by theproduction method of Embodiment 1 in which a TAPS buffer solution isused instead of a HEPES buffer solution.

FIGS. 6A through 6D are cross-sectional views illustrating respectiveprocess steps for fabricating a nonvolatile memory cell according tothis embodiment.

FIG. 7A through 7B show cross-sectional views illustrating the processstep of arranging and immobilizing dots two-dimensionally on the surfaceof a substrate.

FIGS. 8A through 8E illustrate a technique for arranging andimmobilizing complexes two-dimensionally on the surface of a substrate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the method of Douglas et al., a reaction represented by the followingchemical reaction formula 1 is utilized.

As can be understood from the chemical reaction formula 1, as a reactionproceeds, the acidity of a reaction solution is increased. When thereaction solution is at around pH 8, many amino acid residues with anegative charge are exposed on the inside surface of a holding portion 4of apoferritin 1 shown in FIG. 1, and thus Co²⁺ ions are introduced intothe holding portion 4. Because of this, particles of cobalt hydroxideare easily formed in the holding portion 4 of the apoferritin 1.Accordingly, with the pH of the reaction solution kept at around 8, H⁺which is generated as the reaction proceeds is neutralized. Therefore,in the method of Douglas et al., NaOH is added thereto dropwise tocontrol the pH of the apoferritin solution while a cobalt nitratesolution and a hydrogen peroxide solution are gradually added to anapoferritin solution by a very small amount at a time and the solutionis briskly stirred by a stirrer.

As has been described, in the method of Douglas et al., it is difficultto keep the diameter of cobalt particles at the diameter thereof beforethe operations when forming a cobalt-apoferritin complex. The presentinventors, therefore, re-examined the method of Douglas et al. to findthe following problems.

A first problem is in that pH is controlled by not using a buffersolution but by dropping NaOH to an apoferritin solution. The NaOHconcentration abruptly rises in part of the apoferritin solution towhich NaOH is dropped, locally, and then pH is increased. Therefore, thefunction of controlling pH may not be sufficient. Therefore, thedirection of the chemical reaction represented by the reaction formula 1may be reversed, resulting in elution of cobalt particles contained incobalt-apoferritin complexes into the solution.

A second problem is in that NaOH is used. NaOH is a strong proteindenaturant. The NaOH concentration abruptly rises in part of theapoferritin solution to which NaOH is dropped, locally, and thereforeapoferritin may be denatured. Accordingly, apoferritin may be in thestate where it can not fully exhibit its original characteristics. Inother words, apoferritin may not be able to fully hold cobalt particles.

A third problem is in that it is difficult to perform the method on anindustrial scale. In the method of Douglas et al., NaOH is dropped tothe apoferritin solution to control the pH thereof while a cobaltnitrate solution and a hydrogen peroxide solution are gradually added toan apoferritin solution by a very small amount at a time. In thismethod, when the total amount of the reaction solution is about 20-50ml, the chemical reaction can be easily led. However, if a reactionscale is enlarged to an industrial level, it will require a huge amountof time to add cobalt nitrate, a hydrogen peroxide solution and NaOH toan apoferritin solution. Moreover, it will be also difficult touniformly diffuse cobalt nitrate, a hydrogen peroxide solution, and NaOHin a large amount of apoferritin solution. Therefore, the method isconsidered to be not practical.

Embodiment 1

An embodiment of the present invention to be described hereinafter hasbeen devised on the basis of the above-described examination. A methodfor producing a cobalt-apoferritin complex according to this embodimentwill be described with reference to FIGS. 1 through 3.

FIG. 2 is a flowchart illustrating a method for producing acobalt-apoferitin complex according to this embodiment.

First, in Step St1, a reaction solution is prepared by mixing a HEPESbuffer solution, an apoferritin solution and Co²⁺ ion solution (e.g., acobalt nitrate solution) in this order, as shown in FIG. 2.

Next, in Step St2, an oxidizing agent (e.g., H₂O₂) is added to thereaction solution, as shown in FIG. 2. By this operation, cobalthydroxide (CoO(OH)) is introduced into the holding portion 4 of theapoferritin 1 and then cobalt-apoferritin complexes are generated, asshown in FIG. 2.

Note that all of the above-described operations for producing acobalt-apoferritin complex are performed at room temperature or in atemperature range in which a protein is not denatured, while thesolution is stirred by a stirrer.

Next, detail description for each of the steps will be given.

First, in Step St1, the pH of the reaction solution is adjusted to be ina range from about 7.5 to 9.0. More specifically, the pH of the reactionsolution is preferably adjusted to be in a range from about 8.0 to 8.8.When the pH of the reaction solution is in a range from about 8.0 to8.8, many amino acid residues with a negative charge are exposed on theinside surface of the holding portion 4 of the apoferritin 1 and thusCo²⁺ ions are led to the holding portion 4. Therefore, particlescomposed of cobalt hydroxide (CoO(OH)) can be easily formed in theholding portion 4 of the apoferritin 1. The state of cobalt particlesformed at each pH is shown in Table 1.

TABLE 1 Co²⁺ ion concentration pH 8.0 pH 8.2 pH 8.3 pH 8.4 pH 8.6 pH 8.82.0 mM Poor Poor Poor Fair Fair Fair 2.5 mM Not Good Good Good Not Notexamined examined examined 3.0 mM Good Excellent Excellent ExcellentGood Fair 3.5 mM Not Excellent Excellent Excellent Not Not examinedexamined examined 4.0 mM Good Excellent Good Good Poor Poor 5.0 mMExcellent Excellent Not Poor Poor Poor examined

Note that although HEPES is out of the range in which it can exhibit ahigh buffer capacity when the solution is in a pH range from about 8.0to 8.8, the concentration of HEPES may be made to be at a high level.The HEPES concentration in the reaction solution may be at any level aslong as the variation range of pH is sufficiently small, even thoughprecipitates of cobalt hydroxide (CoO(OH)) appear. For example, when theconcentration of cobalt ions in the reaction solution is 3 mM, a HEPESbuffer solution containing HEPES at a concentration of 90 mM or more maybe used.

The concentration of apoferritin in the reaction solution is adjusted tobe in a range from 0.1 to 1 mg/ml (about 0.2-2 μM). More specifically,it is preferably about 0.5 mg/ml (1 μM).

The concentration of Co²⁺ ion is adjusted according to the concentrationof apoferritin. The Co²⁺ ion concentration may be about 1000 to 5000times more than that of the apoferritin concentration. Morespecifically, it is preferably about 2000 to 3000 more than that of theapoferritin concentration. Note that Co²⁺ ions may be added at a higherconcentration than the above-described level. If Co²⁺ ions are added ata high concentration, CoO(OH) is formed vigorously outside of theholding portion of the apoferritin and then cobalt-apoferritin complexesmay be caught by the precipitates. Accordingly, a recovery rate ofcobalt-apoferritin complexes may be reduced.

For example, when the apoferritin concentration is 0.5 mg/ml (about 1μM), Co²⁺ ions are added at a concentration of 2-3 mM. Although anycompound may be used to add Co²⁺ ions, it is particularly preferable touse cobalt ammonium sulfate or cobalt nitrate. If cobalt ammoniumsulfate or cobalt nitrate is used, Co²⁺-HEPES associated pairs whichwill be described later can be easily formed and a reaction will notproceed rapidly (i.e. mixed substances will not react explosively).

Note that in this embodiment, the reaction solution is prepared so thatthe final concentration of HEPES is 30 mM (pH 8.8), the finalconcentration of apoferritin is 0.5 mg/ml (1 μM), and the finalconcentration of Co²⁺ ion is 5 mM in Step St1.

In the Step St2, a hydrogen peroxide solution of 0.01-3% in an equalquantity or a ½ quantity of that of cobalt ion is added to the reactionsolution. For example, when the concentration of Co²⁺ ion is 2 mM, H₂O₂is added to the reaction solution so that the final concentration ofH₂O₂ is in a range from 1 mM to 2 mM.

Note that there may be cases in which apoferritin is denatured byaddition of a hydrogen peroxide solution. Therefore, adding a salt iseffective to stabilize apoferritin. As a salt, for example, Na₂SO₄ canbe used. However, other salts may be also used. Moreover, when acobalt-apoferritin complex is produced, the existence of Cl⁻ ions may bean obstacle to the production (Cl⁻ ions stabilize Co²⁺ ions to inhibitthe formation of CoO(OH)). Therefore, it is preferable to use a salt notincluding Cl.

A salt may be added to the solution at a concentration of 10 mM or more.Test results also show that in the case of Na₂SO₄, it is sufficient forstabilization of apoferritin to add Na₂SO₄ at a concentration of about30-150 mM.

The above-described conditions for the reaction solution will be shownin Table 2.

TABLE 2 Apoferritin Co²⁺ ion HEPES H₂O₂ Quantity 1 2000–500010000–100000 1000–5000 ratio Concentra- 0.5 mg/ml 2 mM to 10 mM to 1 mMto tion (1 μM) 5 nM 100 mM 5 M

It should be avoided that Cl⁻ ions come into the reaction solutionduring the process steps for preparing the reaction solution. Morepreferably, oxygen is exhausted from the reaction solution by bubblingnitrogen or other means.

Under the above-described conditions, the color of the reaction solutionis pink, i.e., a color that Co²⁺ ions exhibit in Step St1.

In Step St2, when CoO(OH) is generated by the addition of a hydrogenperoxide solution, the color of the reaction solution turns to a colorbetween brown and green, i.e., a color that Co³⁺ exhibits. Measured by aspectrophotometer, CoO(OH) has become a hydroxide which has anabsorption peak at around 350 nm.

Normally, if the reaction solution is reacted at room temperature underthe above-described conditions, time required for the reaction is fromseveral hours to several days. However, the temperature of the reactionsolution may be increased to 40° C. to 70° C. to facilitate thereaction. By increasing the solution temperature, the reaction can befinished in several hours or over a night. Since apoferritin particlesbecome unstable at a temperature over 70° C., it is preferable toincrease the temperature of the reaction solution to a temperatureranging from room temperature to 70° C. More specifically, it is morepreferable to increase the temperature of the reaction solution to50-60° C. As another option, if thermophile apoferritin is used, thetemperature may be increased to about 80-100° C. This is because thecrystallinity of generated CoO(OH) under the above-described temperatureconditions is improved in such a case.

Note that in this embodiment, Na₂SO₄ is added to the reaction solutionso that the final concentration of H₂O₂ is 2 mM and the finalconcentration of Na₂SO₄ is 75 mM and the temperature of the reactionsolution is increased to 50° C., in Step St2.

In general, a buffer solution should not influence a chemical substancein a solution. However, in the case of the HEPES buffer solution used inthis embodiment, it is presumed that HEPES and Co²⁺ ions interact witheach other and then form Co²⁺-HEPES associated pairs. The reactionsolution then may be in an equilibrium state represented by thefollowing chemical reaction formula 2.

The state of the reaction solution then is schematically shown in FIG.3.

As the chemical reaction formula 2 and FIG. 3 shows, a Co²⁺-HEPESassociated pair, a simple HEPES ion and a simple Co²⁺ ion are present tomake an equilibrium state.

Assume that used is a reaction solution including water instead of aHEPES buffer solution in Step St1. When a hydrogen peroxide solution isadded to the reaction solution, Co²⁺ ions immediately react, so thatCoO(OH) which is a Co³⁺ compound is formed, as shown in the chemicalreaction formula 1. CoO(OH) is insoluble and thus immediatelyprecipitates. As a result, all Co²⁺ ions become CoO(OH) precipitates.

However, it is also presumed that in this embodiment, when a hydrogenperoxide solution is added, H₂O₂ can not oxidize the Co²⁺-HEPESassociated pairs which are of an associated form of a Co²⁺ ion andHEPES. Therefore, very few Co²⁺ ions existing in the solution areoxidized by H₂O₂ outside of apoferritin, and then become CoO(OH)precipitates.

In contrast, the holding portion 4 of the apoferritin 1 has a negativecharge at around pH 8. Therefore, as shown in FIG. 3, the concentrationof Co²⁺ ion at around pH 8 is higher than that of the outside of theapoferritin 1. Accordingly, CoO(OH) is preferentially formed in theholding portion 4 of the apoferritin 1. Furthermore, contact catalysisoccurs on the surface of CoO(OH) and therefore the reaction isaccelerated rapidly in the holding portion 4.

Since Co²⁺ ions become CoO(OH) precipitates due to the addition of H₂O₂,the number of Co²⁺ ions in the whole solution is reduced. However, sincethe reaction represented by the chemical reaction formula 2 ischemically balanced, the Co²⁺-HEPES associated pairs are dessociated andthus Co²⁺ ions are supplied. Therefore, the concentration of Co²⁺ ion inthe reaction solution is low but can be maintained substantiallyconstant.

As a result of the above-described mechanism, Co²⁺ ions are suppliedfrom the Co²⁺-HEPES associated pairs to the apoferritin 1 and thenconcentrated by the negative charge of holding portion 4 of theapoferritin 1. Thus, the formation of CoO(OH) is facilitated in theholding portion 4 so that a cobalt-apoferritin complex is formed.

Next, operations subsequent to Step St2 will be described.

First, the reaction solution obtained in Step St2 is put into a vesseland is centrifuged at a speed of 3000 rpm for 15-30 minutes by acentrifugal separator such that precipitates are removed. Subsequently,a supernatant liquid obtained after the removal of the precipitates isfurther centrifuged at a speed of 10000 rpm for 30 minutes such that anunnecessary bulk material in which cobalt-apoferritin complexes arecondensed is precipitated and then removed. At this time,cobalt-apoferritin complexes are dispersed in the supernatant liquid.

Next, the solvent of the supernatant is dialyzed to replace a HEPESbuffer solution at pH 7.0 and of 100 mM with an NaCl solution of 150 mM.In this manner, another cobalt-apoferritin complex solution is obtained.It is not particularly necessary to adjust the pH of the solution here.

Subsequently, the cobalt-apoferritin complex solution is condensed bydialysis so that the concentration of cobalt-apoferritin complex withrespect to the whole solution is an arbitrary concentration ranging from1 to 10 mg/l. Thereafter, CdSO₄ is added to the solution so that thefinal concentration of CdSO₄ becomes 10 mM. In this manner,cobalt-apoferritin complexes are condensed.

Next, the cobalt-apoferritin complex solution is centrifuged at a speedof 3000 rpm for 20 minutes so that ferritin aggregates in the solutionare precipitated. Thereafter, the solution is dialyzed such thatbuffering elements in the solution is replaced with a Tris buffersolution at pH 8.0 and of 10-50 containing NaCl of 150 mM.

Next, the cobalt-apoferritin solution is concentrated and then filteredusing a gel filtration column to obtain cobalt-apoferritin complexes.Thereafter, the cobalt-apoferritin complexes are stored in a propersolution.

As has been described, in this embodiment, pH adjustment is performedusing a buffer solution and thereby the pH of the apoferritin solutioncan be set at a desired level. Thus, it is possible to prevent thereaction represented by the chemical reaction formula 1 from proceedingin the reverse direction. Accordingly, elution of cobalt particlescontained in the cobalt-apoferritin complexes into the solution can beprevented. Therefore, in this embodiment, a cobalt-apoferritin complexcontaining a cobalt particle having a uniform diameter can be obtained.

More specifically, NaOH is not used in this embodiment and thusapoferritin is not denatured. Therefore, it is possible to keepapoferritin being in the state of where it can exhibits its originalcharacteristics, i.e., in the state where it can sufficiently holdcobalt particles.

Moreover, in the method for producing a cobalt-apoferritin complexaccording to this embodiment, Co²⁺ ions become CoO(OH) precipitates dueto the addition of H₂O₂ and therefore the number of Co²⁺ ions in thewhole solution is reduced. However, through the parallel reaction of thechemical reaction formula 2, the Co²⁺-HEPES associated pairs aredessociated and Co²⁺ ions are supplied. Co²⁺ ions are condensed becauseof the negative charge of holding portion 4 of the apoferritin 1. In theholding portion 4, formation of CoO(OH) proceeds and cobalt-apoferritincomplexes are formed. Accordingly, even when a reaction scale isincreased to an industrial level, cobalt-apoferritin complexes can beformed only by adding an oxidizing agent without paying specialattentions for uniformly dispersing the oxidizing agent (i.e., H₂O₂ inthis embodiment). Therefore, in the method for producing acobalt-apoferritin complex of this embodiment, the process steps can beperformed in an industrial scale in a relatively simple manner.

FIG. 4 is an electron micrograph of cobalt-apoferritin complexesobtained in this embodiment. In FIG. 4, typical cobalt-apoferritincomplexes in which CoO(OH) is incorporated into (the holding portion of)apoferritin are pointed by the arrows.

Meanwhile, FIG. 5 is an electron micrograph of apoferritin obtained bythe production method of this embodiment in which a phosphoric acidbuffer solution or a TAPS buffer solution is used instead of a HEPESbuffer solution. Now, FIGS. 4 and 5 are compared. In FIG. 5, CoO(OH) isnot incorporated into (the holding portion of) apoferritin as shown bytypical ones pointed by the arrows. More specifically, if a phosphoricacid buffer solution is used, formation of CoO(OH) does not occur in aholding portion of apoferritin. The present inventors also confirmedthat in some other buffer solution (e.g., a Tris buffer solution, a TAPSbuffer solution and an acetic acid buffer solution), formation ofCoO(OH) does not occur in a holding portion of apoferritin. The resultssuggest that all Co²⁺ ions have become CoO(OH) precipitates outside ofapoferritin in the solution and therefore they could not be condensed inthe holding portion. That is to say, it seems that in the case of a TAPSbuffer solution, a Tris buffer solution, a phosphoric acidbuffersolution, an acetic acid buffer solution or the like, there is noequilibrium state like one represented by the chemical reaction formula2 and shown in FIG. 3 between Co²⁺ ions and phosphoric acid, aceticacid, or Tris.

In view of the above described, it is presumed that HEPES used in thisembodiment is a buffer and also functions as an associating agent whichassociates with Co²⁺ ions. Therefore, if different reagents are used asa buffer solution and an associating agent, respectively, instead of theHEPES buffer solution used in this embodiment, a cobalt-apoferritincomplex containing a cobalt particle having a uniform diameter can beobtained as in this embodiment. That is to say, if, for example, a TAPSbuffer solution, a Tris buffer solution, a phosphoric acid buffersolution or the like is used as a buffer solution, and cyclodextrin,crown ether, calix arene or the like is used as an associating agent,the same effects as those of this embodiment can be achieved.

Moreover, in this embodiment, apoferritin is used as a protein forintroducing cobalt. However, other proteins (e.g., Dps protein or CCMVprotein) which can hold metal particles therein may be used instead ofapoferritin.

Furthermore, in this embodiment, H₂O₂ is used as an oxidizing agent.However, other known oxidizing agents may be used. For example, KMnO₄,K₂Cr₂O₇, HNO₃, HClO, or NaClO may be used as an oxidizing agent insteadof H₂O₂.

Embodiment 2

In this embodiment, a nonvolatile memory cell including as a floatinggate a dot body which is formed by utilizing a cobalt-apoferritincomplex produced in Embodiment 1 will be described.

FIGS. 6A through 6D are cross-sectional views illustrating respectiveprocess steps for fabricating a nonvolatile memory cell according tothis embodiment.

First, in the process step shown in FIG. 6A, an isolation oxide film 102is formed on a p-type Si substrate 101 so as to surround an activeregion by LOCOS, and then a gate oxide film 103 that functions as atunnel insulating film is formed on the substrate by thermal oxidation.Thereafter, dot bodies 104 each including a metal or semiconductorparticle having a diameter of about 6 nm are formed on the substrate.The process step of forming the dot bodies 104 will be described later.

Next, in the process step shown in FIG. 6B, a SiO₂ film is deposited onthe substrate by sputtering or CVD so that the dot bodies 104 are buriedin the SiO₂ film.

Next, in the process step shown in FIG. 6C, an Al film is deposited overthe substrate. Subsequently, using a photoresist mask Pr1, the SiO₂ filmand the Al film are patterned to form a silicon oxide film 105 that isto be an inter-electrode insulating film and an Al electrode 106 that isto be a control gate electrode. At this point, part of the gate oxidefilm 103 which is not covered by the photoresist mask Pr1 is removed andthus some of the dot bodies 104 located thereon are also removed at thesame time. Thereafter, using the photoresist mask and the Al electrode106 as a mask, ions of an impurity are injected thereto, thereby formingfirst and second n-type doped layers 107 a and 107 b.

Then, in the process step shown in FIG. 6D, using known techniques, aninterlevel insulating film 108 is formed, a contact hole 109 is formedso as to pass through to the interlevel insulating film 108, the contacthole 109 is filled with tungsten to form a tungsten plug 110, and thenfirst and second aluminum interconnects 111 a and 111 b are formed.

In this embodiment, a p-type Si substrate is used. However, an n-type Sisubstrate may be used. Furthermore, a substrate formed of a GaAscompound semiconductor, some other compound semiconductor, or some othersemiconductor may be used.

Next, in the process step shown in FIG. 6A, a technique for making thedot bodies 104 are formed on the substrate will be described withreference to FIGS. 7 and 8. Note that the present invention is notlimited the following technique, but other known techniques may be alsoused.

In the process step shown in FIG. 7A, cobalt-apoferritin complexes(which will be herein referred to as “complexes”) 150 obtained inEmbodiment 1 are prepared and the complexes 150 are placed on thesurface of a substrate 130. In this manner, a complex film in which thecomplexes 150 are arranged at high density with high precision is formedon the substrate 130. Note that the substrate 130 is a substrateobtained in the process step shown in FIG. 6A, by forming on a p-type Sisubstrate 101 using LOCOS an isolation oxide film 102 which surround anactive region and then forming on the substrate through thermaloxidation a gate oxide film 103 which functions as a tunnel insulatingfilm. In the following description, the substrate 130 is a substrateobtained in the same manner as described here.

Next, in the process step shown in FIG. 7B, protein molecules 140 in thecomplexes 150 are removed so that only cobalt particles 104 a are left.In this manner, the dot bodies 104 are formed on the substrate 130.

A technique for arranging the complexes 150 at high density with highprecision on the surface of the substrate 130 performed in the processstep shown in FIG. 7A, i.e., a method for arranging and immobilizing thecomplexes 150 two-dimensionally on the surface of the substrate 130 willbe described. In this embodiment, the method disclosed in JapanesePatent Publication No. 11-45990 will be described hereinafter withreference to FIG. 8.

First, as shown in FIG. 8A, a liquid 160 in which complexes 150 aredispersed is prepared (i.e., a solution obtained by dispersingcobalt-apoferritin complexes in a solution obtained by mixing in equalvolumes a phosphorus acid buffer solution having a concentration of 40mM and pH 5.3 and a sodium chloride solution having a concentration of40 mM is prepared in this embodiment).

Subsequently, as shown in FIG. 8B, PBLH (poly-1-benzyl-L-histidine) isspread on the surface of the liquid 160 using an injector or the like.In this manner, a polypeptide film 170 of PBLH is formed on the surfaceof the liquid 160. At this time, the pH of the liquid 160 is adjusted.

Next, as shown in FIG. 8C, the complexes 150 adhere to the polypeptidefilm 170 with the lapse of time and then two-dimensional crystals of thecomplexes 150 are formed. This is because the polypeptide film 170 has apositive charge while the complexes 150 have a negative charge.

Next, as shown in FIG. 8D, a substrate 130 is mounted (floated) on thepolypeptide film 170. In this manner, the polypeptide film 170 isadhered to the substrate 130.

Next, as shown in FIG. 8E, the substrate 130 is taken out to obtain thesubstrate 130 to which the two-dimensional crystals of the complexes 150adhere with the polypeptide film 170 interposed therebetween.

Next, further details of the process step shown in FIG. 7B will bedescribed.

Because protein molecules are, in general, heat-sensitive, proteinmolecules of the complexes 150 are removed by heat treatment. Forexample, if the substrate 130 is left to stand for about 1 hour in aninert gas such as nitrogen gas at a temperature of 400-500° C., theprotein molecules 140 and the polypeptide film 170 are burned out andthen cobalt particles 104 a are left on the substrate 130 as dot bodies104 so as to be regularly arranged in a two-dimensional manner at highdensity with high precision.

In the above-described manner, as shown in FIG. 7B, it is possible tomake the cobalt particles 104 a held in the complexes 150 appeartwo-dimensionally on the substrate 130 and also form the dot bodies 104regularly arranged at high density with high precision.

As shown in FIG. 6D, a memory cell 100 according to this embodiment is anonvolatile memory cell which includes an MOS transistor (memorytransistor) having an Al electrode 106 which functions as a controlgate, first and second n-type doped layers 107 a and 107 b whichfunction as a source and a drain, respectively, and utilizes changes inthe threshold voltage of the memory transistor according to the amountof an electric charge stored in the dot bodies 104 each of whichfunctions as a floating gate.

The nonvolatile memory cell can function as a binary memory. However, itis also possible to achieve a multivalued-data storing memory which canstore three- or more-valued data by controlling not only the presence ofcharges to be stored in the dot bodies 104 but also the total amount ofstored charges therein.

When data is erased, FN (Fowler-Nordheim) current passing through anoxide film or direct tunneling current is utilized.

When data is written, FN (Fowler-Nordheim) current passing through anoxide film or a direct tunneling current, or channel hot electron (CHF)injection is utilized

In the nonvolatile memory cell of this embodiment, the floating gate isformed of cobalt particles having a diameter as small as they canfunction as quantum dots and thus the amount of charges to be stored issmall. Therefore, the amount of current flowing when date is written orerased can be reduced, and thus a low-power consumption nonvolatilememory cell can be achieved.

Also, in the nonvolatile memory cell of this embodiment, the diameter ofcobalt particles forming the floating gate is uniform. Thus, thecharacteristics of the cobalt particles when a charge is injected orremoved are also uniform. Therefore, such an operation as injection orremoval of a charge can be controlled in a simple manner.

Therefore, according to the present invention, a cobalt-protein complexcontaining a cobalt particle having a uniform diameter can be obtained.

A method for producing a cobalt-protein complex according to the presentinvention is applicable to fabrication of devices requiring hyperfinepatterning, and more particularly applicable to fabrication of hyperfineelectric devices such as a nonvolatile memory cell including a dot bodyas a floating gate.

1. A method for producing a cobalt-protein complex comprising: the stepa) of preparing a solution including Co²⁺ ions, a protein, and HEPES,and having a pH of not less than 8.0 and not more than 8.8; and a stepb) of adding an oxidizing agent to the solution and thereby making theprotein contain particles composed of cobalt, wherein the protein isapoferritin, the concentration of the Co²⁺ ions is not less than 2.5 mMand not more than 5.0 mM, the pH of the solution is adjusted to be notless than 8.0 and not more than 8.8 when the concentration of the Co²⁺ions is not less than 2.5 mM and not more than 3.5 mM, the pH of thesolution is adjusted to be not less than 8.0 and not more than 8.4 whenthe concentration of the Co²⁺ ions is more than 3.5 mM and not more than4.0 mM, and the pH of the solution is adjusted to be not less than 8.0and not more than 8.2 when the concentration of the Co²⁺ ions is morethan 4.0 mM and not more than 5.0 mM.
 2. The method for producing acobalt-protein complex of claim 1, wherein the oxidizing agent is H₂O₂.3. The method for producing a cobalt-protein complex of claim 1, whereinthe step b) is performed at a temperature of 70° C. or less.
 4. Themethod for producing a cobalt-protein complex of claim 1, wherein theparticles composed of cobalt includes CoO(OH).
 5. The method forproducing a cobalt-protein complex of claim 1, wherein the step b) isperformed at a temperature of not less that 40° C. and not more than 70°C.
 6. The method for producing a cobalt-protein complex of claim 5,wherein the step b) is performed at a temperature of not less than 50°C. and not more than 60° C.